Retroviruses exist as infectious viruses and as endogenous retroviral copies (ERVs) which are viral DNA copies integrated into host DNA. Such ERVs are a permanent part of the host genome and represent 8-10% of host chromosomal DNA. Retroviruses were first recognized as naturally occurring infectious agents linked to various neoplasms in their host species. We have been engaged in an ongoing effort to characterize endogenous and pathogenic viruses, and to identify the various host factors that restrict the replication of these viruses. Mouse leukemia viruses (MLVs) are gammaretroviruses linked to induction of neoplasms and to neurological and immunodeficiency diseases. Inbred strains of laboratory mice and wild mouse species differ in their susceptibility to mouse gammaretrovirus infection and to virus-induced diseases, and they also differ in the types of MLVs that they carry. Susceptibility differences are due to variations in specific host genes that restrict virus replication, and we have been engaged in an ongoing effort to identify and characterize host genes that are either involved in virus resistance or that contribute to the disease process. There are two types of host genes involved in virus-induced disease. First, the mouse genome contains copies of mouse gammaretrovirus genomes, many of which can produce infectious and pathogenic viruses. Second, there are also host factors that interfere directly with virus infection and replication, and we are particularly interested in those factors that inhibit virus entry and post-entry stages of the virus replicative cycle. At the level of entry, resistance can be caused by polymorphisms in the cell surface receptors. After the gammaretrovirus enters the receptive cell, reverse transcription and translocation to the nucleus can be inhibited or altered by virus resistance factors, especially Fv1, mApobec3, and TRIM5alpha. Our group aims to characterize the endogenous retroviruses carried in the mouse genome, and the host encoded resistance factors and their viral targets. Our ultimate goal is to describe co-evolutionary patterns of virus-host interactions in natural populations. This work relies heavily on wild mice because laboratory strains provide only a limited sampling of the genetic diversity in Mus. Also, wild mouse species allow us to examine survival strategies in natural populations that harbor virus and to follow the evolution of the resistance genes. These mice additionally provide a source of novel resistance genes and virus variants. One set of projects aims to characterize endogenous retroviruses (ERVs) of MLVs found in the genomes of mice. These ERVs can produce infectious viruses of three host range subtypes: ecotropic, xenotropic and polytropic mouse leukemia viruses (E-, X-, P-MLVs) which differ in their ability to infect cells of mouse and other species. Leukemogenesis involves generation of recombinants with polytropic host range and these viruses represent recombinants of ERVs derived from all three host range groups. In a previous study we characterized infectious pathogenic and nonpathogenic viral recombinants to identify ERV progenitors and segments that are inked to pathogenesis or host range. More recently, we described the distribution of 45 ERVs in 55 different strains of laboratory mice and analyzed this distribution by common ancestry to describe ERV inheritance patterns among strain lineages, and by incidence of MLV-associated lymphomagenesis. We also typed the same mice for functional variants of the Fv1 restriction gene, a key factor in the expression and spread of MLVs in mice. Our results help define the genetic basis of strain differences in spontaneous lymphomagenesis, describes the distribution of MLV ERVs in strains with shared ancestry, and should help annotate sequenced mouse strain genomes for these insertionally polymorphic and functionally important proviruses. the xenotropic/polytropic subgroups of MLVs use the XPR1 receptor for cell entry, and tropism differences are due to polymorphisms in XPR1 and the viral envelope. To characterize these receptor variants and identify blocks to cross-species transmission, we examined the XPR1 receptors in six mammalian species that restrict different subsets of X/P-MLVs. These restrictive receptors have replacement mutations in regions implicated in receptor function, and some entry restrictions can be relieved by glycosylation inhibitors. Mutation of the cow and hamster XPR1 genes identified a shared, previously unrecognized receptor-critical site. This G/Q503N replacement dramatically improves receptor function. While this substitution introduces an N-linked glycosylation site, XPR1 receptors are not glycosylated indicating that this replacement alters the virus-receptor interface independently of glycosylation. Our data also suggest that an unidentified glycosylated cofactor may influence X/P-MLV entry. Trim5 is a retroviral restriction factor that mediates a post-entry block to infection. Previous studies on the Trim5 locus revealed complicated patterns of gene amplification and the independent birth of gene fusions with CypA in various primate species. However, the evolution of Trim5 in the largest order of mammals, Rodentia, was poorly characterized. We completed an expansive phylogenetic and genomic analysis of the Trim5 cluster in rodents. Our findings reveal substantial evolutionary changes including gene amplifications, rearrangements, loss and fusion. We describe the first independent evolution of TrimCyp fusion genes in rodents. We show that the TrimCyp gene found in the genus Peromyscus was acquired at least 2 million years ago. The TRIMCyp of P. maniculatus can restrict HIV-1 when ectopically expressed, but it does not activate innate immune responses via stimulation of Nf-B or AP-1 promoters. These results describe a complex pattern of differential gene amplification in the Trim5 cluster of rodents and identify the first functional TrimCyp fusion gene outside of primates and tree shrews. Another ongoing study involves characterization of a novel 8.0 kb endogenous retrovirus, XTERV-LS, that we identified in the amphibian, Xenopus tropicalis. This ERV has intact open reading frames for all viral proteins, but has an unusual genomic structure and domain relationships to known retroviruses. Phylogenetic analysis failed to identify close relationships with known retroviruses and XTERV-LS is distinct from the 2 previously described X. tropicalis ERVs: XTERV-LS1 and Xen-1. The reverse transcriptase domain of this ERV shows closest similarity to the ancient envelope (env)-deficient ERV-L family of endogenous retroviruses, and to the exogenous spumaviruses confirming an evolutionary relationship between these subgroups. Finally, we continue to analyze a novel host restriction factor found in most mammals that blocks viral protein production in the later stage of the viral life cycle. Experimental evidence indicates that inspliced message may be the target of this restriction.